Antiviral and Antimicrobial Nucleoside Derivatives: Structural Features and Mechanisms of Action.

The emergence of new viruses and resistant strains of pathogenic microorganisms has become a powerful stimulus in the search for new drugs. Nucleosides are a promising class of natural compounds, and more than a hundred drugs have already been created based on them, including antiviral, antibacterial and antitumor agents. The review considers the structural and functional features and mechanisms of action of known nucleoside analogs with antiviral, antibacterial or antiprotozoal activity. Particular attention is paid to the mechanisms that determine the antiviral effect of nucleoside analogs containing hydrophobic fragments. Depending on the structure and position of the hydrophobic substituent, such nucleosides can either block the process of penetration of viruses into cells or inhibit the stage of genome replication. The mechanisms of inhibition of viral enzymes by compounds of nucleoside and non-nucleoside nature have been compared. The stages of creation of antiparasitic drugs, which are based on the peculiarities of metabolic transformations of nucleosides in humans body and parasites, have been considered. A new approach to the creation of drugs is described, based on the use of prodrugs of modified nucleosides, which, as a result of metabolic processes, are converted into an effective drug directly in the target organ or tissue. This strategy makes it possible to reduce the general toxicity of the drug to humans and to increase the effectiveness of its action on cells infected by the virus.

[Modeling of mechanochemical DNA cleavage by action of ultrasound].

This study offers a simulation of the stretching dynamics of a double-stranded DNA fragment in the high-gradient flow of fluid near collapsing cavitation bubbles. Calculated profiles of elastic tension along the model of a polymer fragment were used to estimate the rates of mechanochemical cleavage at different positions of DNA restriction fragments. The resulting cleavage rate profiles are qualitatively consistent with the experimentally observed profiles of ultrasonic cleavage rates of DNA restriction fragments, which are position-dependent. The proposed model also relates the sequence specificity of ultrasonic DNA cleavage, which was experimentally detected earlier, to the peculiarities of sequence-specific conformational dynamics of ß-D-deoxyribose in the B-form double helix. A quantitative assessment of the ultrasonic DNA cleavage rates for different conformational states of ß-D-deoxyribose derived from the proposed model qualitatively agrees with the experimental data.

[Ultrasonic cleavage of DNA in complexes with Ag(I), Cu(II), Hg(II)].

We investigated a phenomenon of ultrasonic cleavage of DNA complexed with transition metal cations Ag(I), Cu(II) and Hg(II). We found the statistically significant dependence of relative intensity of cleavage on cation type and concentration. Each cation may cause two different types of distortion in the DNA double-helix depending on whether it binds to major or minor DNA groove. The intensity of ultrasonic cleavage decreases if cation binds to the major DNA groove; the intensity of cleavage increases if cation binds to the minor DNA groove and disturbs the hydrogen bonds of complementary base pairs or it intercalates between bases. Both types of DNA distortion can affect the intensity of N-S interconversion of deoxyribose.

The intramolecular impact to the sequence specificity of B-->A transition: low energy conformational variations in AA/TT and GG/CC steps.

It is well known, that local B--> A transformation in DNA is involved in several biological processes. In vitro B<--> A transition is sequence-specific. The physical basis of this specificity is not known yet. Here we analyze the effect of intramolecular interactions on the structural behavior of the GG/CC and AA/TT steps. These steps exemplify sequence specific bias to the B- or A-form structure. Optimization of potential energy of the molecular systems composed of an octanucleotide, neutralized by Na(+) and solvated with TIP3P water molecules in rectangular box with periodic boundary conditions gives the statistically representative sets of low energy structures for GG/CC and AA/TT steps in the middle of the diverse flanking sequences. Permissible 3D variations of GG/CC and AA/TT, and correlation of the relative motion of base pairs in these steps were analyzed. AA/TT step permits high variability for low energy conformers in the B-form DNA and small variability for low energy conformers in the A-form DNA. In contrast GG/CC step permits high variability for low energy conformers in the A-form DNA and small variability for low energy conformers in the B-form DNA. The relative motion of base pairs in GG/CC step is high correlated, while in AA/TT step this correlation is notably less. Atom-atom interactions inside-the-step always favors the B-form and their component - stacking interactions (atom-atom interactions between nucleic bases) is crucial for the duplex stabilization. Formation of the A-form for both steps is a result of interactions with the flanking sequences and water-cation environment in the box. The average energy difference between conformations presenting B-form and A-form for the GG/CC step is high, while for the AA/TT step it is rather low. Thus, intramolecular interactions in GG/CC and AA/TT steps affect the possible conformational diversity ("conformational entropy") of the A- and B- type structures of DNA step. This determines the known bias of the A-form DNA depending on the enrichment of sequences with GG/CC. If structural tuning during the process of protein-DNA complex formation lead to the local B--> A transformation of DNA, it is largely directed by high conformational diversity of GG/CC step in the A-form. In such a case the presence in the target site of both kinds of examined steps ensures the reversible character of ligand binding.

The Intramolecular Impact to the Sequence Specificity of B→A Transition: Low Energy Conformational Variations in AA/TT and GG/CC Steps.

Abstract It is well known, that local B→A transformation in DNA is involved in several biological processes. In vitro B↔A transition is sequence-specific. The physical basis of this specificity is not known yet. Here we analyze the effect of intramolecular interactions on the structural behavior of the GG/CC and AA/TT steps. These steps exemplify sequence specific bias to the B- or A-form structure. Optimization of potential energy of the molecular systems composed of an octanucle-otide, neutralized by Na(+) and solvated with TIP3P water molecules in rectangular box with periodic boundary conditions gives the statistically representative sets of low energy structures for GG/CC and AA/TT steps in the middle of the diverse flanking sequences. Permissible 3D variations of GG/CC and AA/TT, and correlation of the relative motion of base pairs in these steps were analyzed. AA/TT step permits high variability for low energy conformers in the B-form DNA and small variability for low energy conformers in the A-form DNA. In contrast GG/CC step permits high variability for low energy conformers in the A-form DNA and small variability for low energy conformers in the B-form DNA. The relative motion of base pairs in GG/CC step is high correlated, while in AA/TT step this correlation is notably less. Atom-atom interactions inside-the-step always favors the B-form and their component - stacking interactions (atomatom interactions between nucleic bases) is crucial for the duplex stabilization. Formation of the A-form for both steps is a result of interactions with the flanking sequences and water-cation environment in the box. The average energy difference between conformations presenting B-form and A-form for the GG/CC step is high, while for the AA/TT step it is rather low. Thus, intramolecular interactions in GG/CC and AA/TT steps affect the possible conformational diversity ("conformational entropy") of the A- and B- type structures of DNA step. This determines the known bias of the A-form DNA depending on the enrichment of sequences with GG/CC. If structural tuning during the process of protein-DNA complex formation lead to the local B→A transformation of DNA, it is largely directed by high conformational diversity of GG/CC step in the A-form. In such a case the presence in the target site of both kinds of examined steps ensures the reversible character of ligand binding.

Ultrasonic cleavage of nicked DNA.

Structural properties of nicked dsDNA have been an object of numerous studies due to their special role in reparation processes. Here we report experimental results covering ultrasound irradiation of a nicked dsDNA fragments. We have quantitatively estimated ultrasonic cleavage rates in these fragments using the polyacrylamide gel electrophoresis. Data reveal cleavage enhancement in the regions of about 10 b. p. up and down the nick. The intensity of ultrasonic cleavage near the nick is one order of magnitude higher than intensity of ultrasonic cleavage in the same sites of the intact dsDNA fragments. At the same time, the cleavage rates in positions beyond the regions around the nick markedly grow weak comparing to the sequence-specific cleavage rates of intact dsDNA. Thus, the presence of the nick serves as an expressive structural alteration which exceeds any modulation of the structure caused by the base-pair sequence.

[Heterogeneity of double-stranded DNA local structure and dynamics: ultrasound studies].

A method for studying the local inhomogeneities of DNA and its dynamics is proposed. The method is based on the combination of two procedures, splitting the DNA molecules by ultrasound and analysis of DNA fragments obtained by gel electrophoresis. The frequency of cleavage of internucleotide bonds was found to depend on the type of nucleotides forming the bond and on their nearest neighbors. Estimates of cleavage frequencies in each of 16 dinucleotides showed that, in the 5'-d(CpG)-3', 5'-d(CpA)-3', and 5'-d(CpT)-3' dimers, the cleavage occurs considerably more frequently than in the rest, and the frequency of cleavage depends on the nearest neighbors. It was shown that the double-helix cleavage can occur with shifts by several nucleotides. Physical prerequisites were considered that can lead to this pattern of sequence - specific cleavage.

[Theoretical study of the structure of adenosine deaminase complexes with adenosine analogues: I. Aza-, deaza- and isomeric azadeazaanalogues of adenosine].

The conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13 isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme (the 1ADD structure was used) within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of the ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1-deazaadenosine (1ADD) known from the X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of the bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.

[Theoretical study of antagonists and inhibitors of mammalian adenosine deaminase. II. Isomeric aza-deazaanalogues of adenosine]. / Teoreticheskoe issledovanie antagonistov i ingibitorov adenozindezaminazy mlekopitaiushchikh. II. Isomernye aza-dezazaanalogi adenozina.

Isomeric aza-deazaanalogues of adenosine and their N1-protonated forms (except for that of 8-aza-1-deazaadenosine) were studied by computer modeling to find a relationship between their molecular structures and the properties as substrates for the mammalian adenosine deaminase. The atomic charge distribution and maps of the electrostatic potential around their van der Waals molecular surface were calculated using the ab initio STO-3G method. The conformational studies were carried out by the MM+ method of molecular mechanics. The previously proposed mechanism of the substrate acceptance in the active site of mammalian adenosine deaminase was refined, and the potential substrate properties were predicted for two previously unstudied adenosine analogues, 5-aza-9-deazaadenosine and 8-aza-3-deazaadenosine.

[Theoretical study of antagonists and inhibitors of mammalian adenosine deaminase. I. Adenosine and its aza- and deaza-analogs]. / Teoreticheskoe issledovanie antagonistov i ingibitorov adenozindezaminazy mlekopitaiushchikh. I. Adenozin i ego aza- i dezazaanalogi.

Aza- and deazaanalogues of adenosine, including their 1-protonated forms (except for that of 1-deazaadenosine), were studied by computer computation to find a relationship between their molecular structures and substrate properties for the mammalian adenosine deaminase. The atomic charge distribution and maps of the electrostatic potential around their van der Waals molecular surface were calculated for these compounds using the ab initio STO-3G method. The conformational studies were carried out by the MM+ method of molecular mechanics. The mechanism that determines the substrate selectivity of mammalian adenosine deaminase is discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

[Conformational polymorphism and extensibility of DNA quadruplexes formed from d(GT)n repeats]. / Konformatsionnyi polimorfizm i rastiazhimost' kvadrupleksov DNK, obrazovannykh iz d(GT)n povtorov.

We showed earlier that oligonucleotides 3'-d(GT)5-pO(CH2CH2O)3p-d(GT)5-3' form bimolecular quadruplexes with parallel orientation of their strands, which are held by guanine quartets alternating with unpaired thymines (GT quadruplex). This work deals with the conformational polymorphism and extensibility of G quadruplexes in complex with molecules of an intercalating agent ethidium bromide (EtBr). A cooperative mechanism of EtBr binding to the GT quadruplex was revealed. The binding constant K = (3.3 +/- 0.1) x 10(4) M-1, cooperativity coefficient omega = 2.5 +/- 0.2, and maximal amount of EtBr molecules intercalated in GT quadruplex (N = 8) were determined. It was proved experimentally by analysis of adsorption isotherms and theoretically by mathematical modeling that the GT quadruplex is capable of double extension, which is indicative of the high elasticity of this four-stranded helix. Two most stable conformations of GT quadruplexes with thymine residues intercalated and/or turned outside were found by mechanico-mathematical modeling. The equilibrium is shifted toward the conformation with the looped out thymine residues upon intercalation of EtBr molecules into the GT quadruplex.

Parallel purine-pyrimidine-purine triplex: experimental evidence for existence.

Oligonucleotides 5'-d(CT)5-L-d(AG)5-L-d(GA)5-3' and 5'-d(GA)5-L-d(TC)5-L-d(GA)5-3' [L = pO(CH2CH2O)3p] were studied by thermal denaturation, chemical modification and binding of fluorescent dyes. Both oligonucleotides are shown to fold back on itself twice forming at pH 7 a sufficiently stable triplex ether with antiparallel-oriented oligopurine strands (the first compound) or parallel-oriented oligopurine strands (the second compounds). The parallel triplex is significantly less stable than the antiparallel one. On the basis of conformational modeling, possible types of base tripling in the triplets are proposed. Thus our data provide the first convincingly evidence for the existence of a purine-pyrimidine-purine triplex with parallel orientation of identical strands.

The R-form of DNA does exist.

Oligonucleotide 5'-d(CATGCTAACT)-L-d(AGTTAGCATG)-L-d(CATGCTAACT)-3' [L = pO(CH2CH2O)3p] is shown to fold back on itself twice forming at pH 7 a sufficiently stable triplex (Tm is about 30 degrees C) with parallel-orientated identical strands (the recombinant or R-form of DNA). Experimental evidence was obtained by studying thermal denaturation, chemical modification and binding of fluorescent probes. The stability of the R-triplex increases in the presence of divalent ions or spermidine. Its structure is characterized by a certain heterogeneity that causes the cooperativity of a triplex-to-duplex transition to decrease. On the basis of conformational modeling, the possible types of base tripling in all four triplets are proposed. The experimental data as well as the molecular mechanic calculations indicate that the stabilities of triplets in the R-triplex decrease in the order: G:C-G = A:T-A >> T:A-T > C:G-C.

Three-stranded clip of the oligonucleotide 5'-(dT)10pO(CH2CH2O)3p(dT)10pO(CH2CH2O)3p(dA)10-3'.

Temperature dependence of UV and CD spectra of the oligonucleotide 5'-(dT)10-L-(dT)10-L-(dA)10-3' [tripl(ATT)] [L = -pO(CH2CH2O)3p-] in phosphate buffer, pH 7, at various NaCl concentrations and in the presence or absence of 0.01 M MgCl2 has been studied. At low oligonucleotide concentrations (2.2 x 10(-5) M nucleotide concentration) all structural transitions proceed intramolecularly. Tripl(ATT) exists in three forms: as a three-stranded clip (at low temperatures), a double-stranded hairpin (at intermediate temperatures), and as an open strand (at high temperatures). Thermodynamic parameters of the triplex formation depending on the NaCl concentration were calculated. The CD spectra were assigned to the single-, double-, and three-stranded forms. Ethidium bromide (EtBr) binding to the three-stranded clip was studied. Ethidium bromide molecules were shown to intercalate into the triple helix with the stable complex formation (association constant is 10(6)). One molecule of three-stranded clip binds not more than three EtBr molecules. The proposed synthetic model (oligonucleotide blocks coupled by hydroxyalkyl chains) has been shown to be convenient for studies of the physical and chemical properties of the triplex and other multistranded complexes of DNA.

[A triple-stranded "clip" from the oligonucleotide 3'-(dA)10-pO(Ch2Ch2O)3p-(dT)10-pO(CH2CH2O)3-p-(dT)10(-5)]. / Trekhtsepochechnaia "skrepka" iz oligonukleotida 3'-(dA)10-pO(CH2CH2O)3p-(dT)10-pO(CH2CH2O)3p-(dT)10(-5).

The temperature dependence of the UV- and CD-spectra of the oligonucleotides 3'-d(A)10-L-(T)10-5' [anti(AT)], 3'-d(A)10-L-d(T)10-3' [par(AT)] and 3'-d(A)10-L-(dT)10-L-(dT)10-5' [tripl(ATT)] (L = -PO(CH2CH2O) 3p-) in the phosphate buffer at pH 7 under different concentrations of NaCL and in the presence or absence of 0.01 M MgCl2 was studied. All registered structural changes are the result of intramolecular processes if the concentrations of the oligonucleotides is low (about 2.2.10(-5) M). Par(AT) and anti(AT) exist in the only two forms, transforming into each other: under low temperatures they exist as hairpins with the parallel or antiparallel orientation of chains accordingly which transform into unfolded chains when the temperature increased. In contrast trip(ATT) exists in the three different forms depending on the temperature and ion conditions. They are: the three- stranded clip, the two-stranded hairpin with a single stranded "tail" and completely unfolded chain. For the first time this work presents thermodynamic parameters of the triplex formation from deoxyoligonucleotides depending on NaCl concentration. We have registered the CD spectra to one-, two-, and three-stranded forms. Ethidium bromide binding to three-stranded "clip" was investigated, and it was established that molecules of the dye may intercalate into the "clip" with formation of stable complexes (the constant of association 10(6) M-1). It is maximum three molecules of ethidium bromid which may bound to one molecule of the three-stranded clip. It has been shown that the suggested synthetic model (three oligonucleotide blocks combined by hydroxyalkyl chains) is the most convenient for physico-chemical investigations of triplexes today.

[Four-stranded complexes of oligonucleotides--quadruplexes]. / Chetyrekhtsepochechnye kompleksy oligonukleotidov--kvadrupleky.

The review presents analysis of the experimental, model and calculation studies concerned with the formation of the four-stranded helices of the natural and synthetic oligonucleotides. Guanine-rich oligonucleotides form stable four-stranded helices. Structures of such complexes were investigated by means of X-rays and spectrographic methods. These works have been reviewed in the first part. There are three possible variants of noncanonical structures formed by oligoguanylic acids. Two of them--four-stranded helices differed by the mutual direction of the sugar-phosphate chains. The third one is the two-stranded hairpin. Regulation of the number of cellular processes by means of the structural conversions between these three forms of guanine-rich motifs are investigated in articles reviewed in the second part. These works are concerned with the structural organization and functions of telomers, and on the other hand with the possible role of quadruplexes in self-recognition processes of the four homologous chromatids during meiosis and the following recombination. The third part of the review considers quadruplexes with an arbitrary sequence. In general there are model works inspired by investigations of recombination and replication processes. Experimental data concerned with the formation of quadruplex structures from two decamer Watson-Crick base paired duplexes oligo(dA).oligo(dT) are also presented.